Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain

2022 | journal article. A publication with affiliation to the University of Göttingen.

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​Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain​
Liu, T.; Stephan, T.; Chen, P.; Keller-Findeisen, J.; Chen, J.; Riedel, D. & Yang, Z. et al.​ (2022) 
Proceedings of the National Academy of Sciences119(52) art. e2215799119​.​ DOI: https://doi.org/10.1073/pnas.2215799119 

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Authors
Liu, Tianyan; Stephan, Till; Chen, Peng; Keller-Findeisen, Jan; Chen, Jingting; Riedel, Dietmar; Yang, Zhongtian; Jakobs, Stefan ; Chen, Zhixing
Abstract
Capturing mitochondria’s intricate and dynamic structure poses a daunting challenge for optical nanoscopy. Different labeling strategies have been demonstrated for live-cell stimulated emission depletion (STED) microscopy of mitochondria, but orthogonal strategies are yet to be established, and image acquisition has suffered either from photodamage to the organelles or from rapid photobleaching. Therefore, live-cell nanoscopy of mitochondria has been largely restricted to two-dimensional (2D) single-color recordings of cancer cells. Here, by conjugation of cyclooctatetraene (COT) to a benzo-fused cyanine dye, we report a mitochondrial inner membrane (IM) fluorescent marker, PK Mito Orange (PKMO), featuring efficient STED at 775 nm, strong photostability, and markedly reduced phototoxicity. PKMO enables super-resolution (SR) recordings of IM dynamics for extended periods in immortalized mammalian cell lines, primary cells, and organoids. Photostability and reduced phototoxicity of PKMO open the door to live-cell three-dimensional (3D) STED nanoscopy of mitochondria for 3D analysis of the convoluted IM. PKMO is optically orthogonal with green and far-red markers, allowing multiplexed recordings of mitochondria using commercial STED microscopes. Using multi-color STED microscopy, we demonstrate that imaging with PKMO can capture interactions of mitochondria with different cellular components such as the endoplasmic reticulum (ER) or the cytoskeleton, Bcl-2-associated X protein (BAX)-induced apoptotic process, or crista phenotypes in genetically modified cells, all at sub-100 nm resolution. Thereby, this work offers a versatile tool for studying mitochondrial IM architecture and dynamics in a multiplexed manner.
Issue Date
2022
Journal
Proceedings of the National Academy of Sciences 
Project
EXC 2067: Multiscale Bioimaging 
TRR 274: Checkpoints of Central Nervous System Recovery 
TRR 274 | Z01: Bioimaging Platform 
FOR 2848: Architektur und Heterogenität der inneren mitochondrialen Membran auf der Nanoskala 
FOR 2848 | Z01: Elektronenmikroskopie 
Working Group
RG Jakobs (Structure and Dynamics of Mitochondria) 
RG Riedel 
ISSN
0027-8424
eISSN
1091-6490
Language
English
Sponsor
Beijing Municipal Science and Technology Commission http://dx.doi.org/10.13039/501100009592
Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659
EC | European Research Council http://dx.doi.org/10.13039/501100000781

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